This invention relates to the genetic engineering of cells and to beer brewing.
Technology currently exists for introducing heterologous (i.e., modified or foreign) genes into laboratory strains of yeast of the genus Saccharomyces, particularly S. cerevisiae. Two types of plasmid vectors have been used for this purpose, replicating and integrating. Replicating vectors contain an origin of DNA replication that functions in yeast, so that the plasmid is maintained extrachromosomally, as a circular episome. Integrating vectors do not contain such an origin and therefore require insertion into a yeast chromosome to be stably maintained.
Both types of plasmids can be introduced into yeast cells by standard transformation methods. Since successful uptake and establishment of plasmid DNA by competent yeast cells is a relatively rare event (&lt;10.sup.-3), a selection mechanism is required to allow identification of transformants.
Most commonly, selection is accomplished by introducing auxotrophic mutations into the recipient yeast strain. The commonly used mutations are ura3, leu2, trp1, and his3. The plasmid of interest bears a wild type copy of one of these genes. Since the wild type copy on the plasmid is dominant to the host chromosomal allele, selection for cells that receive the plasmid is easily accomplished on a minimal medium lacking the nutrient that is required by the auxotrophic host cell.
There have also been reports of the use of antibiotic resistance to select transformed cells. Replicating vectors have been described that are based on the sensitivity of most Saccharomyces strains to the commercially available neomycin analog, antibiotic G418. Jimenez et al. (1980) Nature 287, 869; Hollenberg (1982) In Current Topics in Microbiology and Immunology, Hofschneider et al., eds. (Springer-Verlag NY); Webster et al. (1983) Gene 26, 243. Webster et al. also describe an integrating plasmid vector which could not be directly selected for by resistance to G418. These vectors contain a gene, called kan.sup.r, neo.sup.r, or G418.sup.r from the bacterial transposon Tn903, and a yeast origin of replication; the bacterial gene is preceded by its native bacterial promoter.
Another replicating vector has been described which contains the gene for resistance to the antibiotic hygromycin B under the control of a yeast promoter. Gritz et al. (1983) Gene 25, 178.
Beer brewing using yeasts, e.g., members of the genus Saccharomyces, requires the presence of mono-, di-, or tri-saccharides in the fermentation culture medium ("wort"), which the yeasts metabolize in the production of ethanol, CO.sub.2 and other metabolites. After yeast fermentation, starches and complex oligosaccharides (those larger than three glucose units) remain soluble but unmetabolized. These oligosaccharides, which are flavorless and colorless, add only to the caloric content of beer.
The production of low starch ("light") beer requires removal of some of the unmetabolized soluble starch and complex oligosaccharides present in the wort that normally remain in the beer after fermentation by yeast. Several methods have been used to reduce the content of starch and complex oligosaccharides in low calorie beer:
1) Passing the wort over an immobilized enzyme, glucoamylase, which is capable of breaking down starch and complex oligosaccharides. PA1 2) Addition of soluble glucoamylase to the wort prior to or during fermentation. PA1 3) Prolonging the mashing process, during which endogenous barley amylases degrade starch. PA1 4) Adding malt flour to the wort during fermentation. PA1 5) Substituting fermentable sugars, such as corn syrup, for various amounts of the starch derived from cereal grains. PA1 6) Diluting the final product with water.